Fabrication and evaluation of biomimetic biodegradable tissue-engineered annulus fibrosus scaffold / 中国组织工程研究

BACKGROUND: It is still difficult to construct tissue-engineered anulus fibrosus scaffolds which have bionic structure, suitable biodegradability and good biocompatibility. OBJECTIVE: To fabricate bionic biodegradable scaffolds with polycaprolactone (PCL) and polydioxanone (PDS) and evaluate the feasibility as a tissue-engineered annulus fibrosus scaffold. METHODS: Five groups of scaffolds at different PCL/PDS proportions were prepared by melt spinning technique: PCL, PCL/PDS70/30, PCL/PDS50/50, PCL/PDS30/70, and PDS groups. Scanning electron microscopy was used to observe the structure and measure the fiber diameter and pore size of these prepared scaffolds. The mechanical properties and contact angle of the scaffolds were measured. The in vitro and in vivo biodegradation of the scaffolds were observation by in vitro simulation and subcutaneous implantation. The expression of inflammatory factors interleukin-1β and tumor necrosis factor-α in the biodegraded tissues was detected. Human Wharton’s jelly mesenchymal stem cells were cultured for 7 days. Cell viability and proliferation was determined by live/dead cell staining. This study was approved by the Medical Ethics Committee of Tianjin Hospital, China on March 2, 2016. RESULTS AND CONCLUSION: Scanning electron microscopy results showed that the thickness of the scaffold fibers was uniform and the angle between fibers was 60°. The mechanical properties analysis showed that the tensile and compressive modulus of the PDS group was the lowest, which did not meet the mechanical requirements of the anulus fibrosus; the tensile and compressive modulus in the PCL group was the highest, and those in the PCL/PDS70/30 and PCL/PDS50/50 group were moderate. Hydrophilicity test showed that higher PDS proportion led to better hydrophilicity. Biodegradation test showed that the biodegradation of pure PDS and PCL/PDS30/70 was too fast, that of PCL was too slow, and that of PCL/PDS70/30 and PCL/PDS50/50 was appropriate. Analysis of inflammatory response around the biodegraded tissue showed that higher proportion of PCL in the scaffold resulted in more severe inflammatory response. CCK-8 and live/dead cell staining showed that human Wharton’s jelly mesenchymal stem cells had good proliferative activity and high survival rate in the PCL/PDS70/30, PCL/PDS50/50, and PCL/PDS30/70 groups. These results suggest that scaffolds in the PCL/PDS70/30 and PCL/PDS50/50 groups can simulate the structure of natural annulus fibrosus, have appropriate biodegradability, excellent mechanical properties and good biocompatibility, which make it a suitable candidate for tissue-engineered annulus fibrosus scaffold.

The response of cyclic tensile strain on the BMSCs co-cultured human degenerative anulus fibrosus cells / 天津医药

Objective To investigate the effects of different cyclic tensile strains on the proliferation and expression of bone marrow stromal cells (BMSCs)-cocultured human degenerated anulus fibrosus (AF) cells. Methods AF cells were isolated from a patient with degenerated intervertebral disc degeneration (IVD), which were co-cultured with BMSCs. The solely cultured AF cells were used as control group. The two groups of cells were expanded in monolayer, and cyclically strained for 3 hours, which were applied 0, 5%, 10%, 15%and 20%strains at a frequency of 0.25 Hz using BioDynamic test instrument. A flow cytometry method was used to examine the AF cell proliferation at 24 hours followed the application of cyclic tensile strains. After the total RNA was extracted, real-time PCR technology was used to detect the gene expression of collagenⅠand aggrecan. Results Under the same appropriate stress, the proliferative index (PI), the proportion of cells in the period of DNA synthesis, the expression of collagenⅠand aggrecan were significantly higher in the co-cultured group than those of control group (P<0.05). However, the best mechanical stimulation was different in the two groups. For the AF cells, the peaks of PI, the proportion of cells in the period of S period, the expression of collagenⅠand aggrecan were found in the 10% strain group, while for the co-cultured cells, they were found in the 15% strain group. Conclusion Co-culturing with BMSCs has a positive effect on the proliferation and expression of human degenerative fibrous ring cells, which can protect AF cells from bad stress stimulation.

Response of cyclic tensile strain on the human degenerative anulus fibrosus cells / 国际生物医学工程杂志

Objective To investigate the effects of different cyclic tensile strain on the proliferation of human anulus fibrosus cells from degenerated discs.Methods Anulus fibrosus(AF) cells were isolated from a degenerated human IVD,expanded in monolayer,and cyclically strained for 3 hours,applying 0,5％,10％,15％ and 20％ strains at a frequency of 0.25 Hz with the use of the DioDynamic test instrument.The flow cytometry method was used to examine the Af cells proliferation at 24 hours following application of the cyclic tensile strains.Results The proliferative index (PI) increased with the magnitude value of cyclic tensile strain except 20％ group.The most significant increase of proliferation index were found in 15％ group.Conclusion There might be some corelationships between magnitude of cyclic tensile strain and the proliferation of the degenerative AF cells.

Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

OBJECTIVE: Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. METHODS: Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-1beta, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-1beta, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. CONCLUSION: We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Histological Composition of the Extruded Intervertebral Disc in Lumbar Intervertebral Disc Herniation / 체질인류학회지

The aim of this study was to investigate histological composition (ratio of anulus fibrosus, cartilage endplate, and nucleus pulposus) of the herniated lumbar intervertebral disc fragments. The relative percentage of anulus fibrosus, cartilage endplate, and nucleus pulposus was examined in the herniated interverterbral disc fragments. The herniated intervertebral discs took out from the 31 patients were stained with H-E. The stained tissues were photographed, and the histological composition of the herniated intervertebral discs was analyzed after marking 3 components on the photograph with microscopic observation. The composition of the herniated lumbar intervertebral disc is 50.7% of nucleus pulposus, 41.3% of anulus fibrosus, and 8.0% of cartilage endplate. The proportion of the endplate in the herniated intervertebral disc in male was tend to be higher than that in female, and the proportion of the nucleus pulposus was tend to be decreased with aging, however, fail to reach statistical significance. This result may provide fundamental information for accessing clinical symptoms including pain in lumbar vertebral disc herniation.

Structural Changes of the Nucleus Pulposus Cells and Cartilage Cells of Age-Related Intervertebral Disc in Mongrel Dog / 대한해부학회지

The intervertebral discs of the dog from the newborn, the 1 year old, and the 5 years old were examined about the cell morphologies and their structural changes according to the advancement of age by histomorphometry and electron microscopy. The three regions of the disc - the nucleus pulposus, the inner and outer annulus fibrosus were considered. Each region was divided into the three zones - the peripheral, intermediate, and central zones. The cell morphology of the three regions were as follows : A. The nucleus pulposus 1. At the newborn, the cells of the nucleus pulposus had long cytoplasmic processes forming large extracellular vacuoles. In the peripheral and intermediate zones, the cells with euchromatic nucleus had fine cytoplasmic granule-like fibers and some of free ribosomes. In the central zone, the cells with heterochromatic nucleus had many cytoplasmic vacuoles filled with free ribosomes. 2. At the 1 years old, the cell-group composed of 4 to 8 cells contacted each other with their cytoplasmic processes. In the peripheral zone, the elliptical chondroblast-like cells with euchromatic nucleus had well-developed rough endoplasmic reticulum and mitochondria. In the intermediate zone, the foam-like large round cells with euchromaic nucleus had multiple cytoplasmic vacuoles filled with granular contents. In the central zone, the cells with heterochromatic nucleus had large cytoplasmic vacuoles, and seemed to be degenerated type of the outer zone-cells. 3. At the 5 years old, the cells in the periphery were rhomboid or polygonal with euchromatic nucleus, and had broad cytoplasmic contact surface with adjacent cells. They had small cytoplasmic vacuoles, well-developed Golgi apparatus and rough endoplasmic reticulum. The cells in the intermediate zone had heterochromatic nucleus and a lot of variable size of cytoplasmic vacuoles, and seemed to be degenerated form of the cells of the periphery. In the central zone, the star-like cells with heterochromatic nucleus had multiple sharp cytoplasmic processes and vacant cytoplasm because of large cytoplasmic vacuoles. B. The cartilaginous part and the inner annulus fibrosus 1. At the newborn, the outer zone of the cartilaginous part of the intervertebral disc showed the isogenous group with 2 to 3 chondrocytes within the cell nest. The cells were distributed mainly around the blood vessels, and had euchromatic nucleus. They had small cytoplasmic vacuoles, well-developed rough endoplasmic reticulum and abundant ribosomes. The cells of the inner zone showed heterochromatic nucleus, well-developed rough endoplasmic reticulum and expanding terminal cisternae. 2. At the 1 year old, the inner annulus fibrosus was divided into the fibrocartilaginous part and the hyaline cartilaginous part. The former, the outer zone showed the cell nest with 2 to 4 of elliptical chondrocytes which had euchromatic nucleus, small cytoplasmic vacuoles and scattered fine granules. The latter, the inner zone showed the chondrocytes within the dissociated cell nest with heterochromatic nucleus. They had disperse rough endoplasmic reticulum and microvilli-like cytoplasmic processes. 3. At the 5 years old, the annulus fibrosus was composed mainly of fibrocartilage. The chondrocyte showed heterochromatic nucleus, long cytoplasmic processes, large cytoplasmic vacuoles and densely packed abundant granules. C. The outer annulus fibrosus 1. At the 1 year old, the outer annulus fibrosus was composed of the palisading collagenous bundles. The chondrocytes with heterochromatic nucleus located in the cartilage cell nest. The cells had well-developed rough endoplasmic reticulum, mitochondria, and Golgi apparatus, and 4 to 5 processes of interterritorial matrix surrounded the collagenous bundles. 2. At the 5 years old, the collagenous bundles were invaded by the processes of interterritorial matrix, and made them incomplete ones. The cell had heterochromatic nucleus and scanty cytoplasm containing small mitochondria and poorly developed organelles. The Summary of The Above Mentioned Findings Are : 1. The cells of the nucleus pulposus were degenerated gradually from the periphery toward the center. 2. The cells of the nucleus pulposus were degenerated gradually according to the aging process. 3. With age, the cells of the annulus fibrosus were degenerated, the hyaline cartilage was replaced gradually by fibrocartilages, and fragmentation of the collagenous bundles appeared. 4. The older the age, the smaller the nucleus of the nucleus pulposus cells, and the larger the nucleus of the annulus fibrosus cells were encountered in the histomorphometric measurement.